title: The generation of olfactory epithelial neurospheres in vitro predicts engraftment capacity following transplantation in vivo.
abstract: The stem and progenitor cells of the olfactory epithelium maintain the tissue throughout life and effectuate epithelial reconstitution after injury. We have utilized free-floating olfactory neurosphere cultures to study factors influencing proliferation, differentiation, and transplantation potency of sphere-grown cells as a first step toward using them for therapeutic purposes. Olfactory neurospheres form best and expand most when grown from neonatal epithelium, although methyl bromide-injured or normal adult material is weakly spherogenic. The spheres contain the full range of epithelial cell types as marked by cytokeratins, neuron-specific antigens, E-cadherin, Sox2, and Sox9. Globose basal cells are also prominent constituents. Medium conditioned by growth of phorbol ester-stimulated, immortalized lamina propria-derived cells (LP(Imm)) significantly increases the percentage of Neurog1eGFP(+) progenitors and immature neurons in spheres. Sphere-forming capacity resides within selected populations; FACS-purified, Neurog1eGFP(+) cells were poorly spherogenic, while preparations from ΔSox2eGFP transgenic mice that are enriched for Sox2(+) basal cells formed spheres very efficiently. Finally, we compared the potency following transplantation of cells grown in spheres vs. cells derived from adherent cultures. The sphere-derived cells engrafted and produced colonies with multiple cell types that incorporated into and resembled host epithelium; cells from adherent cultures did not. Furthermore, cells from spheres grown in conditioned media from the phorbol ester-activated LP(Imm) line gave rise to significantly more neurons after transplantation as compared with control. The current findings demonstrate that sphere formation serves as a biomarker for engraftment capacity and multipotency of olfactory progenitors, which are requirements for their eventual translational use.
authors.affiliation: Department of Anatomy and Cellular Biology, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111, USA.
authors.forename: Richard C
authors.initials: RC
authors.lastname: Krolewski
authors.forename: Woochan
authors.initials: W
authors.lastname: Jang
authors.forename: James E
authors.initials: JE
authors.lastname: Schwob
mesh_terms: D000818:Animals; D049109:Cell Proliferation; D005434:Flow Cytometry; D007150:Immunohistochemistry; D051379:Mice; D008822:Mice, Transgenic; D009831:Olfactory Mucosa / Q000166:cytology* / Q000378:metabolism / Q000637:transplantation*; D018034:Olfactory Receptor Neurons / Q000166:cytology* / Q000378:metabolism / Q000637:transplantation*; D033581:Stem Cell Transplantation; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism